The present invention relates to receptors for glial cell line-derived neurotrophic factor (GDNF) and provides nucleic acid and amino acid sequences encoding GDNF receptor (GDNFR). The present invention also relates to therapeutic techniques for the treatment of GDNF-responsive conditions.
Glial Cell Line-Derived Neurotrophic Factor
Glial cell line-derived neurotrophic factor (GDNF) was initially isolated and cloned from rat B49 cells as a potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons (Lin et al., Science, 260, 1130-1132, 1993). Recent studies have indicated that this molecule exhibits a variety of other biological activities, having effects on several types of neurons from both the central and peripheral nervous systems. In the central nervous system (CNS), GDNF has been shown to prevent the axotomy-induced death of mammalian facial and spinal cord motor neurons (Li et al., Proceedings Of The National Academy Of Sciences, U.S.A., 92, 9771-9775, 1995; Oppenheim et al., Nature, 373, 344-346, 1995; Yan et al., Nature, 373, 341-344, 1995; Henderson et al., Science, 266, 1062-1064, 1994; Zurn et al., Neuroreport, 6, 113-118, 1994), and to rescue developing avian motor neurons from natural programmed cell death (Oppenheim et al., 1995 supra). Local administration of GDNF has been shown to protect nigral dopaminergic neurons from axotomy-induced (Kearns and Gash, Brain Research, 672, 104-111, 1995; Beck et al., Nature, 373, 339-341, 1995) or neurotoxin-induced degeneration (Sauer et al., Proceedings Of The National Academy Of Sciences U.S.A., 92, 8935-8939, 1995; Tomac et al., Nature, 373, 335-339, 1995). In addition, local administration of GDNF has been shown to induce sprouting from dopaminergic neurons, increase levels of dopamine, noradrenaline, and serotonin, and improve motor behavior (Tomac et al., 1995 supra).
More recently, GDNF has been reported to be a potential trophic factor for brain noradrenergic neurons and Purkinje cells. Grafting of fibroblasts ectopically expressing GDNF prevented 6-hydroxydopamine-induced degeneration and promoted the phenotype of adult noradrenergic neurons in vivo (Arenas et al., Neuron, 15, 1465-1473, 1995), while exogeneously applied GDNF effectively promoted survival and morphological differentiation of embryonic Purkinje cells in vitro (Mount et al., Proceedings Of The National Academy Of Sciences U.S.A., 92, 9092-9096, 1995). In the peripheral nervous system, GDNF has been shown to promote the survival of neurons in nodose, ciliary, and sympathetic ganglia, as well as small populations of embryonic sensory neurons in dorsal root ganglia (DRG) and trigeminal ganglia (Trupp et al., Journal Of Cell Biology, 130, 137-148, 1995; Ebendal et al., Journal Of Neuroscience Research, 40, 276-284, 1995; Oppenheim et al., 1995 supra; Yan et al., 1995 supra; Henderson et al., 1994 supra). GDNF has also been reported to enhance the expression of vasoactive intestinal peptide and preprotachykinin-A mRNA in cultured superior cervical ganglion (SCG) neurons, and thus effects the phenotype of SCG neurons and induces bundle-like sprouting (Trupp et al., 1995 supra).
Expression of GDNF has been observed in a number of different cell types and structures of the nervous system. In the CNS, GDNF mRNA expression has been observed by reverse transcriptase polymerase chain reaction (RT-PCR) in both developing and adult rat striatum, the major target of nigral dopaminergic innervation, and widely in other regions, including hippocampus, cortex, thalamus, septum, cerebellum, spinal cord, and medulla oblongata (Arenas et al., supra 1995; Poulsen et al., Neuron, 13, 1245-1252, 1994; Springer et al., Experimental Neurology, 127, 167-170, 1994; Stroemberg et al., Experimental Neurology, 124, 401-412, 1993; Schaar et al., Experimental Neurology, 124, 368-371, 1993). In human, GDNF transcripts have also been detected in striatum, with highest level in the caudate and lower levels in the putamen. Detectable levels are also found in hippocampus, cortex, and spinal cord, but not in cerebellum (Schaar et al., Experimental Neurology, 130, 387-393, 1994; Springer et al., 1994 supra). In the periphery, GDNF mRNA expression has been reported in DRG and SCG of postnatal day 1 rats, sciatic nerve, and primary cultures of neonatal Schwann cells (Trupp et al., 1995 supra; Hoffer et al., Neuroscience Letters, 182, 107-111, 1994; Henderson et al., 1994 supra; Springer et al., 1994 supra). In addition, recent studies have shown that GDNF transcripts are also widely expressed in peripheral non-neuronal organs, including postnatal testis and kidney, embryonic whisker pad, stomach, and skin. Expression can be detected at lower levels in embryonic muscle, adrenal gland and limb bud, and in postnatal lung, liver and ovary (Trupp et al., 1995 supra; Henderson et al., 1994 supra). So far, however, the biological significance of the non-neuronal expression of GDNF is not clear.
Detailed descriptions of the preparation and characterization of GDNF protein products may be found in U.S. patent application Ser. No. 08/182,183 filed May 23, 1994 and its parent applications (also see PCT/US92/07888, WO 93/06116 filed Sep. 17, 1992 and European Patent Application No. 92921022.7, Publication No. EP 610 254) the disclosures of which are hereby incorporated by reference. Additional GDNF protein products are described in pending U.S. patent application Ser. No. 08/535,681 filed Sep. 28, 1995, the disclosure of which is hereby incorporated by reference. As used herein, the term xe2x80x9cGDNF protein productxe2x80x9d includes biologically active synthetic or recombinant GDNF proteins and analogs, as well as chemically modified derivatives thereof. GDNF analogs include deletion variants such as truncated GDNF proteins, as well as insertion and substitution variants of GDNF. Also included are GDNF proteins that are substantially homologous to the human GDNF protein.
GDNF Therapy
GDNF therapy is helpful in the treatment of nerve damage caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells. Such nerve damage may occur from a wide variety of different causes. Nerve damage may occur to one or more types of nerve cells by: (1) physical injury, which causes the degeneration of the axonal processes and/or nerve cell bodies near the site of injury; (2) temporary or permanent cessation of blood flow to parts of the nervous system, as in stroke; (3) intentional or accidental exposure to neurotoxins, such as chemotherapeutic agents (e.g., cisplatinum) for the treatment of cancer or dideoxycytidine (ddC) for the treatment of AIDS; (4) chronic metabolic diseases, such as diabetes or renal dysfunction; or (5) neurodegenerative diseases such as Parkinson""s disease, Alzheimer""s disease, and amyotrophic lateral sclerosis (ALS), which result from the degeneration of specific neuronal populations.
Several studies indicate that GDNF therapy is particularly helpful in the treatment of neurodegenerative conditions such as the degeneration of the dopaminergic neurons of the substantia nigra in Parkinson""s disease. The only current treatments for Parkinson""s disease are palliative, aiming at increasing dopamine levels in the striatum. The expected impact of GDNF therapy is not simply to produce an increase in the dopaminergic neurotransmission at the dopaminergic nerve terminals in the striatum (which will result in a relief of the symptoms), but also to slow down, or even stop, the progression of the degenerative processes and to repair the damaged nigrostriatal pathway and restore its function. GDNF may also be used in treating other forms of damage to or improper function of dopaminergic nerve cells in human patients. Such damage or malfunction may occur in schizophrenia and other forms of psychosis. The only current treatments for such conditions are symptomatic and require drugs which act upon dopamine receptors or dopamine uptake sites, consistent with the view that the improper functioning of the dopaminergic neurons which innervate these receptor-bearing neuronal populations may be involved in the disease process.
Receptors
A number of receptors which mediate binding and response to protein factors have been characterized and molecularly cloned, including receptors for insulin, platelet derived growth factor, epidermal growth factor and its relatives, the fibroblast growth factors, various interleukins, hematopoietic growth factors and ciliary neurotrophic factor (U.S. Pat. No. 5,426,177). Study results indicate that some receptors can bind to multiple (related) growth factors, while in other cases the same factor can bind and activate multiple (related) receptors (e.g., Lupu et al., Science, 249:1552-1555, 1990; Dionne et al., EMBO J., 9:2685-2692, 1990; Miki et al., Science, 251:72-75, 1991). Most receptors can broadly be characterized as having an extracellular portion or domain responsible for specifically binding a protein factor, a transmembrane domain which spans the cell membrane, and an intracellular domain that is often involved in initiating signal transduction upon binding of the protein factor to the receptor""s extracellular portion. Although many receptors are comprised of a single polypeptide chain, other receptors apparently require two or more separate subunits in order to bind to their protein factor with high-affinity and to allow functional response following binding (e.g., Hempstead et al., Science, 243:373-375, 1989; Hibi et al., Cell, 63:1149-1157, 1990).
The extracellular and intracellular portions of a given receptor may share common structural motifs with the corresponding regions of other receptors, suggesting evolutionary and functional relationships between different receptors. These relationships can often be quite distant and may simply reflect the repeated use of certain general domain structures. For example, a variety of different receptors that bind unrelated factors make use of xe2x80x9cimmunoglobulinxe2x80x9d domains in their extracellular portions, while other receptors utilize xe2x80x9ccytokine receptorxe2x80x9d domains in their factor-binding regions (e.g., Akira et al., The FASEB J., 4:2860-2867, 1990). A large number of receptors with distinct extracellular binding domains (which thus bind different factors) contain related intracytoplasmic domains encoding tyrosine-specific protein kinases that are activated in response to factor binding (e.g., Ullrich and Schlessinger, Cell, 61:203-212, 1990). The mechanisms by which factor-binding xe2x80x9cactivatesxe2x80x9d the signal transduction process is poorly understood, even in the case of receptor tyrosine kinases. For other receptors, in which the intracellular domain encodes a domain of unknown function or in which the binding component associates with a second protein of unknown function (e.g., Hibi et al., Cell, 63:1149-1157, 1990), activation of signal transduction is not well characterized.
The mode of action of GDNF in vivo is not clearly elucidated in the art, in part due to the absence of information on a receptor for GDNF. Two groups have independently found that striatum injected [125I]-labeled GDNF can be retrogradely transported by dopaminergic neurons in the substantia nigra (Tomac et al., Proceedings Of The National Academy Of Sciences Of The United States Of America. 92, 8274-8278, 1995; Yan et al., 1995 supra). Retrograde transport of [125I]-GDNF by spinal cord motor neurons, DRG sensory neurons and neurons in the B layer of retina ganglia was also been observed. These retrograde transport phenomena can all be specifically inhibited by 100-fold or higher concentrations of unlabeled GDNF, suggesting a saturable, receptor-mediated transport process. In vitro, recombinant GDNF has been shown to enhance the survival and promote dopamine uptake of cultured dopaminergic neurons at very low concentrations. The observed half-maximal effective concentration (EC50) of GDNF on these neurons is 0.2 to 1.6 pM (Lin et al., 1993 supra). GDNF has also been shown to support the survival of dissociated motor neurons at low concentrations. The reported EC50 of GDNF on motor neurons, in a 5 to 10 fM range, is even lower than that on dopaminergic neurons (Henderson et al., 1994 supra).
Taken together, these observations indicate that receptor(s) for GDNF expressed in these cells have very high ligand binding affinities. Similar to members of the TGF-xcex2 family, the widely diversified tissue distribution and varied biological function of GDNF on different populations of cells suggest that different types of receptor(s) for GDNF or receptor complexes may exist. Saturation steady-state and competitive binding of [125I]-GDNF to E10 chick sympathetic neurons has shown that these neurons express GDNF binding sites differing from those observed in dopaminergic and motor neurons. The half maximal saturation concentration and the half-maximal inhibition concentration of GDNF on these binding sites is in the range of 1 to 5 nM (Trupp et al., 1995 supra). Similarly, the EC50 of GDNF in supporting the survival of sympathetic neurons from P1 rat SCG has also been reported to be in the nanomolar range (Trupp et al., 1995 supra).
To better understand the mechanism by which GDNF activates signal transduction to exert its affects on cells, it would be beneficial to identify the receptor(s) which mediate binding and response to this protein factor. It would also be beneficial for GDNF therapy to identify and make possible the production of accessory molecules which provide for or enhance GDNF signal transduction. Moreover, the identification of a protein receptor for GDNF would provide powerful applications in diagnostic uses, for example, as an aid in determining if individuals would benefit from GDNF protein therapy. Furthermore, the protein receptor for GDNF could be a key component in an assay for identifying additional molecules which bind to the receptor and result in desired biological activity.
The present invention provides nucleic acid sequences which encode a neurotrophic factor receptor protein having an amino acid sequence as depicted in FIGS. 2 and 4 (SEQ. ID. NOs.: 2 and 4) as well as biologically equivalent analogs. The neurotrophic factor receptor protein and protein products of the present invention are designated herein as glial cell line-derived neurotrophic factor receptor (GDNFR) protein and protein products. The novel GDNFRs are functionally characterized by the ability to bind GDNF specifically and with high affinity, and to act as part of a molecular complex which mediates or enhances the signal transduction affects of GDNF. GDNFR protein products are typically provided as a soluble receptor protein and in a substantially purified form.
In one aspect, the present invention provides for the production of GDNFR protein products by recombinant genetic engineering techniques. In an alternative embodiment, the GDNFR proteins are synthesized by chemical techniques, or a combination of the recombinant and chemical techniques.
In another aspect of the present invention, the GDNFR proteins may be made in glycosylated or non-glycosylated forms. Derivatives of GDNFR protein typically involve attaching the GDNFR protein to a water soluble polymer. For example, the GDNFR protein may be conjugated to one or more polyethylene glycol molecules to decrease the precipitation of the GDNFR protein product in an aqueous environment.
Yet another aspect of the present invention includes the various polynucleotides encoding GDNFR proteins. These nucleic acid sequences are used in the expression of GDNFR in a eukaryotic or prokaryotic host cell, wherein the expression product or a derivative thereof is characterized by the ability to bind to GDNF and thereby form a complex capable of mediating GDNF activity, such as increasing dopamine uptake by dopaminergic cells. The polynucleotides may also be used in cell therapy or gene therapy applications. Suitable nucleic acid sequences include those specifically depicted in the Figures as well as degenerate sequences, naturally occurring allelic variations and modified sequences based on the present invention.
Exemplary nucleic acid sequences include sequences encoding a neurotrophic factor receptor protein comprising an amino acid sequence as depicted in FIGS. 2 and 4 (SEQ ID NOs. 2 and 4) capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and mediating cell response to GDNF, and biologically equivalent analogs thereof. Such sequences include: (a) a sequence set forth in FIG. 1 (SEQ ID NO. 1) comprising nucleotides encoding Met1 through Ser465 or FIG. 3 (SEQ ID NO. 3) comprising nucleotides encoding Met1 through ger468 encoding a neurotrophic factor receptor (GDNFR) capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and mediating cell response to GDNF; (b) a nucleic acid sequence which (1) hybridizes to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity; and (c) a nucleic acid sequence which but for the degeneracy of the genetic code would hybridize to a complementary sequence of (a) and (2) encodes an amino acid sequence with GDNFR activity. Also disclosed herein are vectors such nucleic acid sequences wherein the sequences typically are operatively linked to one or more operational elements capable of effecting the amplification or expression of the nucleic acid sequence. Host cells containing such vectors are also contemplated. Typically, the host cell is selected from mammalian cells and bacterial cells, such as a COS-7 cell or E. coli, respectively.
A further aspect of the present invention involves vectors containing the polynucleotides encoding GDNFR proteins operatively linked to amplification and/or expression control sequences. Both prokaryotic and eukaryotic host cells may be stably transformed or transfected with such vectors to express GDNFR proteins. The present invention further includes the recombinant production of a GDNFR protein wherein such transformed or transfected host cells are grown in a suitable nutrient medium, and the GDNFR expressed by the cells is, optionally, isolated from the host cells and/or the nutrient medium. The present invention further includes the use of polynucleotides encoding GDNFR and vectors containing such polynucleotides in gene therapy or cell therapy.
The host cell may also be selected for its suitability to human implantation, wherein the implanted cell expresses and secretes a neurotrophic factor receptor of the present invention. The host cell also may be enclosed in a semipermeable membrane suitable for human implantation. The host cell may be transformed or transfected ex vivo. An exemplary device for treating nerve damage involves: (a) a semipermeable membrane suitable for implantation; and (b) cells encapsulated within the membrane, wherein the cells express and secrete a neurotrophic factor receptor as disclosed herein. The membrane is selected from a material that is permeable to the neurotrophic factor receptor protein but impermeable to materials detrimental to the encapsulated cells.
Methods for the recombinant production of a neurotrophic factor receptor of the present invention are also disclosed. An exemplary methods involves: (a) culturing a host cell containing a nucleic acid sequence encoding a neurotrophic factor receptor of the present invention, such as an amino acid sequence depicted in FIGS. 2 and 4 (SEQ ID NOs. 2 and 4) capable of completing with glial cell line-derived neurotrophic factor (GDNF) and mediating cell response to GDNF, or biologically equivalent analogs thereof; (b) maintaining said host cell under conditions suitable for the expression of said neurotrophic factor receptor by said host cell; and (c) optionally, isolating said neurotrophic factor receptor expressed by said host cell. The host cell may be a prokaryotic cell or a eukaryotic cell. If bacterial expression is involved, the method may further include the step of refolding the neurotrophic factor receptor.
The present invention includes an isolated and purified protein comprising an amino acid sequence as depicted in FIGS. 2 and 4 (SEQ ID NOs. 2 and 4) capable of complexing with glial cell line-derived neurotrophic factor (GDNF) and mediating cell response to GDNF, and biologically equivalent analogs thereof. Exemplary analogs include, but are not limited to, proteins comprising the amino acid sequence Ser18 through Pro446, Asp25 through Leu447 and Cys29 through Cys442 as depicted in FIG. 2 (SEQ. ID. NO:2) as well as proteins comprising the amino acid sequence Met17 through Pro449 and Cys29 through Cys443 as depicted in FIG. 4 (SEQ. ID. NO:4). The proteins of the present invention may be glycosylated or non-glycosylated and may be produced by recombinant technology or chemical synthesis. The present invention further includes nucleic acid sequences encoding a receptor protein comprising such amino acid sequences.
Also disclosed herein are pharmaceutical compositions comprising a protein receptor of the present invention in combination with a pharmaceutically acceptable carrier. A variety of other formulation materials may be used to facilitate manufacture, storage, handling, delivery and/or efficacy.
Another aspect of the present invention includes the therapeutic use of GDNFR genes and proteins. For example, a circulating or soluble GDNFR protein product may be used alone or in conjunction with GDNF in treating disease of or injury to the nervous system by enhancing the activity of transmembrane signaling of GDNF. Thus, the proteins and pharmaceutical compositions of the present invention may be used in treating improperly functioning dopaminergic nerve cells, Parkinson""s disease, Alzheimer""s disease and amyotrophic lateral sclerosis. Alternatively, a recombinant GDNFR gene may be inserted in the cells of tissues which would benefit from increased sensitivity to GDNF, such as motor neurons in patients suffering from amyotrophic lateral sclerosis. In yet another embodiment, GDNFR may be used to block GDNF activity in cases where GDNF activity is thought to be detrimental. The GDNFR may be used to verify that observed effects of GDNF are due to the GDNFR.
In another aspect of the invention, GDNFR probes may be used to identify cells and tissues which are responsive to GDNF in normal or diseased states. Alternatively, the probes may be used to detect an aberrancy of GDNFR expression in a patient suffering from a GDNF-related disorder.
In a further aspect of the invention, GDNFR probes, including nucleic acid as well as antibody probes, may be used to identify GDNFR-related molecules. For example, the present invention provides for such molecules which form a complex with GDNFR and thereby participate in GDNFR function. As another example, the present invention provides for receptor molecules which are homologous or cross-reactive antigenically, but not identical to GDNFR.
The present invention also provides for the development of both binding and functional assays for GDNF based on the receptor. For example, assay systems for detecting GDNF activity may involve cells which express high levels of GDNFR, and which are therefore extremely sensitive to even very low concentrations of GDNF or GDNF-like molecules. In yet another embodiment, soluble GDNFR may be used to bind or detect the presence of GDNF or GDNF-like molecules.
In addition, the present invention provides for experimental model systems for studying the physiological role of GDNF. Such systems include assays involving anti-GDNFR antibodies or oligonucleotide probes as well as animal models, such as transgenic animals which express high levels of GDNFR and therefore are hypersensitive to GDNF or animals derived using embryonic stem cell technology in which the endogenous GDNFR genes were deleted from the genome. An anti-GDNFR antibody will binds a peptide portion of the neurotrophic factor receptor proteins. Antibodies include monoclonal and polyclonal antibodies. Alternatively, immunological tags for which antibodies already exist may be attached to the GDNFR protein to aid in detection. Such tags include but are not limited to Flag (IBI/Eastman Kodak) and myc sequences. Other tag sequences such as polyhistidine have also been used for detection and purification on metal chelating columns.
Additional aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following description, which details the practice of the present invention.